Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase

Abstract

Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarse-grained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding.

Figure 1

Schematic of the refolding experiments for I91₃-Luciferase-I91₄ construct and I91₉. The distance between the substrate and the tip is modulated by the piezo, which executes a constant velocity ramp as shown in (A). The force on the molecule as determined by the extension, closely approximated by a wormlike chain, is shown in (B). The shaded region in (A) and (B) is where the molecule experiences a force that is below the typical isometric unfolding force (5 pN). The physical schematic of the experiment is shown in (C), where the piezo moves toward the cantilever, at which a protein can fold (denoted by the star), which is determined by the subsequent unfolding that results in a force peak.

Figure 2

Four examples of successive refolding attempts on a single molecule of I91₃-Luciferase-I91₄. The order of recordings goes from bottom to top for each set. As can be seen, the I91 molecules (200 pN unfolding force) are able to refold successfully upon each attempt. A single non-I91 peak appears a fraction of the time, and a wormlike chain (dashed line) shows a contour-length increments that match previously reported contour-length increments for individual domains of Luciferase.

Figure 3

Distributions of the contour-length increments for non-I91 peaks in refolding recordings (peaks with force <120 pN; see Fig. S9 for all peaks). Top shows the distribution for refolding of I91₉. Bottom shows the distribution for the refolding of I91₃-Luciferase-I91₄, which has events not present in I91₉ and match previous results for the unfolding of full Luciferase. To see this figure in color, go online.

Figure 4

Representation of the main folding routes from coarse-grain simulations (back arrows representing unfolding not shown; for complete Markov state model, see Fig. S2). The numbers above each state represent that state number for the 14-state clustering (reference in Fig. S3), while the percentage is the probability of obtaining that structure during any given folding pathway (see Materials and Methods). The arrow thickness qualitatively represents the relative flux through the pair of states. Structures with a star indicate that they were evaluated experimentally by SMFS. To see this figure in color, go online.

Figure 5

Representative structures of the topologically misfolded states obtained during folding of Luciferase, with the state labeled in bold (referring to 14-state clustering in Fig. S2). The structures are colored from red (N-terminal residues) to blue (C-terminal residues). The right side shows a template molecule of the correctly folded form of Luciferase. To see this figure in color, go online.

Figure 6

Three representative examples of experimental force-spectroscopy recordings obtained for each construct in this study. All the vertical scale bars represent 100 pN and the horizontal scale bars all represent 50 nm. Arrows indicate the force rupture event used in analysis.

Figure 7

Experimental SMFS results of the contour-length increment (left) and unfolding force (right) for Luciferase truncations. Previous results on the full Luciferase (47) are shown at the top for comparison, which shows unfolding of C-terminal domain (light shading), middle domain (dark shading), and N-terminal domain (solid). The dashed bar shows the result for the contour-length increment calculated from SMD simulations.